The use of a biothreatening agent in 2001 in the United States and the ready availability of a variety of such agents highlight the need for rapid, reliable diagnostics capable of detecting and identifying these dangerous pathogens early in infection and of monitoring disease progression and therapeutic efficacy. The long-term goal of this project is to develop a platform tool based on surface enhanced Raman scattering (SERS) microscopy for the simultaneous rapid detection and identification of a broad range of category A-C priority bacterial pathogens. The strategy employed in this project is to build a simple, sensitive diagnostic platform that requires no labeling, hybridization, growth, or PCR, but relies on the enormous Raman scattering signal enhancements due to a unique aggregated gold particle-covered SiO2 substrate. Detailed spectra of single bacterial cells have been obtained on this substrate with signal acquisition times of about 20 seconds. The SERS microscopic diagnostic platform will provide rapid, reagentless, specific identification of species within minutes, and function as a portable device for field use. The following specific aims will be accomplished in Phase I: (1) development of rapid, robust methods of enriching bacteria from clinical samples for SERS; (2) optimization of the SERS substrate performance; and (3) development and implementation of a quantitative algorithm to validate the diagnostic specificity and sensitivity of SERS for identification of B. anthracis in both sterile and non-sterile body fluids. In Phase II, the SERS platform will be transformed into an instrument for clinical use. The capabilities of the diagnostic will be expanded to include (a) other priority category A-C pathogens, (b) a large reference database of SERS spectra, and (c) a portable detection instrument capable of automated chip scanning and bacterial enrichment by microfluidics.